The goal of this project is to determine structure in the HIV fusion protein gp41 to better understand the initial process of HIV infection at the level of viral membrane fusion. Gp41 is embedded in the viral envelope and upon activation, undergoes conformational changes which direct fusion between viral envelope and the target cell membrane to initiate a new cycle of infection. Present structures of fusion active gp41 lack the critical membrane-binding fusion peptide (FP) region as well as the relevant membrane environment, and a major gap in our understanding of gp41 fusion function centers on our limited knowledge of the role of the fusion peptide. We propose to probe the structure of the FP region of gp41 folded in the low energy hairpin (FP-hairpin), which is a late stage gp41 fusion conformation. Our interdisciplinary approach involves peptide synthesis, protein expression, isotopic labeling, native chemical ligation, experimental NMR spectroscopy of polypeptides and proteins in membranes, protein crystallization, and X-ray diffraction analysis. Specifically, we will apply solid-state NMR (SSNMR) techniques to resolve FP backbone structure of site-specific, isotopically (15N and 13C) labeled FP-hairpin interacting with membranes. Full structure will be determined based on X-ray diffraction of unlabeled FP-hairpin crystallized from detergent. In addition, we propose to determine structure for part of an earlier gp41 fusion conformation termed the pre-hairpin intermediate (PHI) in membranes. A construct named N70 reproduces structural organization of the PHI, and SSNMR techniques will be applied to determine backbone structure and tertiary organization in the FP and coiled-coil regions of 13C and 15N labeled N70 in membranes. A fragment which represents primarily the FP region, termed FP34, will be analogously labeled and analyzed in membranes using SSNMR and compared to N70 and FP-hairpin measurements to determine if it serves as a suitable model to reproduce gp41 FP structure. The following SSNMR analyses will be applied: (i) Local secondary structure will be probed with chemical shift measurements of site specific 13C and 15N labeled residues using rotational-echo double-resonance (REDOR) filtering. Intermolecular 13C-13C and 13C-15N distances, based on dipolar coupling measurement using both REDOR and finite-pulse constant-time double-quantum build-up (fpCTDQBU) techniques, will detail tertiary organization and provide atomic resolution structural information. PUBLIC HEALTH RELEVANCE A clear, high resolution structural model of the HIV fusion peptide in context of its gp41 fusion protein has broad implications by detailing a potential therapeutic target to inhibit fusion and therefore infection, and by providing functional insights into the mechanism by which viral fusion proteins merge membranes.